No description available.

No description available.

No description available.

No description available.

Glycerol-3-phosphate cytidylyltransferase from 𝘚𝘵𝘢𝘱𝘩𝘺𝘭𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘢𝘶𝘳𝘦𝘶𝘴 complexed with CTP (TarDₛₐ-CTP) was crystallized by the hanging drop-vapor diffusion method at 22°C. Determination of crystallization condition included examination of the amount of precipitant, investigation of the effects of small molecules, and alteration of the rate of diffusion. With these three optimization steps, crystals suitable for x-ray diffraction study were produced. During data processing, TarDₛₐ-CTP was determined to belong to the space group P3₁21, with unit-cell dimensions a=b=92.2 and c=156.1Å. The crystal structure of TarDₛₐ-CTP was solved to 3.0Å by molecular replacement, using TagD from 𝘉𝘢𝘤𝘪𝘭𝘭𝘶𝘴 𝘴𝘶𝘣𝘵𝘪𝘭𝘪𝘴 as a search model. Unlike the search model, TarDₛₐ appears as a tetramer in the asymmetric unit. This result also confirms the gel-filtration and ultracentrifugation studies that were done previously. Although TarDₛₐ crystals were grown in the presence of CTP, the crystal structure does not reveal convincing data for the location and position of this co-factor. However, the data suggests a possible location for CTP in one of the four subunits in an orientation that differs from that of TagD_Bₛ. Unfortunately, the resolution of this data set at 3.0Å is not high enough to corroborate this finding. / Thesis / Master of Science (MS)

No description available.

The antimicrobial effects of silver nanoparticles were studied. Silver nanoparticles were synthesized through wet chemistry method, and were dispersed in aqueous suspension. With nanoscale silica particles served as heterogeneous nucleation sites, silver nanoparticles were formed anchoring on the silica surface. Suspensions were found to be stable at high silver concentrations as well as over a broad pH range. By varying the processing conditions, diameter of the silver nanoparticles could be controlled between ~2 nm to ~25 nm. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to reveal the formation and the corresponding morphology of the silver-silica coupling nanoparticles. Ultra-violet visible (UV-vis) scanning spectrophotometer was used to detecting the distinct absorption spectrum of silver nanoparticles. The antimicrobial activities of these silver-silica coupling nanoparticles were investigated. E. coli and S aureus were used as representatives of Gram-negative and Gram-positive bacteria, respectively. Bacteriological tests showed either bacterial growth inhibition or cell death occurred, corresponding to different concentrations of silver nanoparticles and the type of bacteria that were testing on. Fluorescent microscopic images were also provided to confirm the bacterial viability after several hours' treatment with silver nanoparticles. / Master of Science

Staphylococcus aureus is a Gram-positive bacterium and part of the human bacterial microflora but it also is an opportunistic pathogen and a notorious cause of hospital – acquired and epidemic infections. S. aureus shows a remarkable adaptation to a range of niches within its human host.Previously viewed as an exclusively extracellular pathogen, S. aureus has been demonstrated to replicate inside virtually all cell types after cell invasion. S. aureus thereby either multiplies within phagosomal compartments in professional phagocytes or escapes into the cytosol prior to replication in non-professional phagocytic cells such as epithelial cells. Besides α-type phenol soluble modulins(PSMα, an important role in phagosomal escape was attributed to the non-ribosomal peptide synthase (NRPS) AusAB. AusAB incorporates the aromatic amino acids (AAAs) phenylalanine or tyrosine, as well as the branched-chain amino acids (BCAAs) valine and leucine into three cyclic dipeptides collectively called aureusimines: Phevalin, Tyrvalin and Leuvalin. The role of AusAB in S. aureus infection is not entirely understood. BCAAs are essential amino acids for S. aureus and serve as protein building blocks,precursors for the biosynthesis of branched-chain fatty acids, as well as regulatory molecules for the transcriptional regulator CodY. Severe BCAA depletion triggers stringent response. It is therefore counterintuitive that AusAB would incorporate valine into aureusimines thereby depleting BCAAs in host niches where available nutrients are scarce. The present study therefore analysed the role of the AusAB NRPS and its BCAA-derived monoketopiperazine products, in the interaction of S. aureus with various host niches and with other bacterial species. By using genetic tools and metabolomic approaches, it could be established that the AusAB NRPS preferentially incorporates only the exogenous aromatic amino acids phenylalanine and tyrosine, while the source of valine can be either endogenous or exogenous. By using promoter reporter assays, an effect of the global SaeR regulator on ausAB expression was observed. Additionally, a possible role of the NRPS in modulatory metabolic processes of overflow metabolism emerged. Finally, while the role of AusAB in infection is still unclear, experiments using ex vivo human lung tissue suggest, for the first time, that AusAB NRPS might be involved in the staphylococcal virulence against human lung tissue. By employing bioluminescence and biofilm assays in both Gram-negative (Vibrio harveyi and Pseudomonas aeruginosa) and Gram-positive (CoNS Staphylococcus spp.) model species, a potential role as a Quorum Quenching molecule arose for Phevalin, but not Tyrvalin. While studying a potential connection of AusAB and stringent response, an unexpected role for the BrnQ1 BCAA transporter was revealed. BrnQ1 is known as the major BCAA import protein in S. aureus. The bacteria therefore rely on its activity in environments with low concentrations of available peptides or free BCAAs. The present study shows that BrnQ1 is necessary for efficient phagosomal escape of S. aureus in epithelial cells and that BrnQ1-mediated BCAA uptake is crucial for intracellular bacterial replication in epithelial cells, macrophages and whole human blood. Moreover, BrnQ1 loss of function allows bacteria to survive indefinitely inside macrophages without causing phenotypic changes in colony morphology, size and pigmentation as well as haemolysis after recovery. A dual RNA-sequencing approach further showed that, while no major host transcriptomic rearrangements occurred in human macrophages infected with brnQ1 mutants compared to wild-type S. aureus, intracellular brnQ1 mutants do not respond to local host iron depletion and thus fail to upregulate iron uptake systems. In summary, the present work elaborates on multiple functions of BCAA-based small molecules as well as BCAA-transporters in the S. aureus host and bacterial interactions. This work provides a consolidation of the role of the AusAB NRPS in S. aureus lung infection, gives an insight into the unique mode of action of the NRPS and attributes a novel function for the NRPS-product Phevalin in bacterial communication providing, to my knowledge, the first example of a natural monoketopiperazine involved in bacterial communication. Further, experiments using ex vivo human lung tissue suggest, for the first time, that AusAB NRPS might be involved in the staphylococcal virulence against human lung tissue. Additionally, this work describes a potential mechanism of S. aureus long-term survival in macrophages involving a BCAA-iron metabolism axis. / Staphylococcus aureus ist ein Gram-positives Bakterium und Teil der menschlichen bakteriellen Mikroflora, aber auch ein opportunistisches Pathogen und eine allgemein bekannte Ursache für Krankenhaus- und epidemische Infektionen. S. aureus zeigt eine bemerkenswerte Anpassung an eine Reihe von Nischen innerhalb seines menschlichen Wirtes. Früher wurde der Erreger ausschließlich als extrazelluläres Pathogen angesehen. Doch es hat sich gezeigt, dass S. aureus nach einer Zellinvasion in praktisch allen Zelltypen replizieren kann. Dabei vermehrt sich S. aureus in professionellen Phagozyten innerhalb phagosomaler Kompartimente, in nicht-professionellen phagozytischen Zellen wie Epithelzellen entweicht das Bakterium vor der Replikation in das Zytosol. Neben α-Typ Phenollöslichen Modulinen (PSMα) wurde der nicht-ribosomalen Peptidsynthase (NRPS) AusAB eine wichtige Rolle beim phagosomalen Ausbruch zugeschrieben. AusAB baut die aromatischen Aminosäuren (AAAs) Phenylalanin oder Tyrosin, sowie die verzweigtkettigen Aminosäuren (BCAAs) Valin und Leucin zu den drei zyklische Dipeptiden Phevalin, Tyrvalin und Leuvalin zusammen, die unter dem Namen Aureusimine zusammengefasst werden. Die Rolle von AusAB bei der S. aureus-Infektion ist nicht vollständig geklärt. BCAAs sind essentielle Aminosäuren für S. aureus und dienen als Proteinbausteine, Vorstufen für die Biosynthese von verzweigtkettigen Fettsäuren sowie als regulatorische Moleküle für den Transkriptionsregulator CodY. Eine starke BCAA-Depletion löst eine stringente Antwort aus. Es ist daher kontraintuitiv, dass AusAB Valin in Aureusimine einbaut und damit BCAAs in Wirtsnischen, in denen die verfügbaren Nährstoffe knapp sind, dezimiert. In der vorliegenden Studie wurde daher die Rolle der AusAB NRPS und seiner BCAA-abgeleiteten Monoketopiperazin-Produkte bei der Interaktion von S. aureus mit verschiedenen Wirtsnischen und mit anderen Bakterienarten analysiert. Durch den Einsatz von genetischen Werkzeugen und metabolomischen Ansätzen konnte festgestellt werden, dass die AusAB NRPS bevorzugt nur die exogenen aromatischen Aminosäuren Phenylalanin und Tyrosin einbaut, während die Quelle für Valin entweder endogen oder exogen sein kann. Mit Hilfe von Promotor-Reporter-Assays wurde ein Effekt des globalen Regulators SaeR auf die ausAB-Expression beobachtet. Zusätzlich zeigte sich eine mögliche Rolle des NRPS bei modulatorischen Stoffwechselvorgängen des Überlaufmetabolismus. Schließlich, während die Rolle von AusAB bei der Infektion noch unklar ist, deuten Experimente mit ex vivo menschlichem Lungengewebe zum ersten Mal darauf hin, dass das NRPS von AusAB an der Virulenz von Staphylokokken gegenüber menschlichem Lungengewebe beteiligt sein könnte. Durch den Einsatz von Biolumineszenz- und Biofilm-Experimenten bei repräsentativen Gramnegativen (Vibrio harveyi und Pseudomonas aeruginosa) als auch Gram-positiven (CoNS Staphylococcus spp.) Spezies ergab sich für Phevalin, nicht aber für Tyrvalin, eine mögliche Rolle als Quorum Quenching-Molekül. Bei Untersuchung einer möglichen Verbindung von AusAB und der stringenten Antwort wurde eine unerwartete Rolle für den BrnQ1 BCAA-Transporter entdeckt. BrnQ1 ist als das wichtigste BCAAImportprotein in S. aureus bekannt. Die Bakterien sind daher auf seine Aktivität in Umgebungen mit niedrigen Konzentrationen an verfügbaren Peptiden oder freien BCAAs angewiesen. Die vorliegende Studie zeigt, dass BrnQ1 für einen effizienten phagosomalen Ausbruch von S. aureus in Epithelzellen notwendig ist und dass die BrnQ1-vermittelte BCAA-Aufnahme für die intrazelluläre bakterielle Replikation in Epithelzellen, Makrophagen und menschlichem Vollblut entscheidend ist. Darüber hinaus ermöglicht der Funktionsverlust von BrnQ1 den Bakterien ein unbegrenztes Überleben im Inneren von Makrophagen ohne phänotypische Veränderungen in Koloniemorphologie, -größe und -pigmentierung sowie in Hämolyse nach der Rückgewinnung der Bakterien. Ein dualer RNASequenzierungsansatz zeigte darüber hinaus, dass in humanen Makrophagen, die mit brnQ1- Mutanten infiziert waren, im Vergleich zu Wildtyp S. aureus keine größeren Transkriptomänderungen im Wirt auftraten. Jedoch reagierten intrazelluläre brnQ1-Mutanten nicht auf eine lokale Eisenverarmung des Wirts und regulieren somit keine Eisenaufnahmesysteme hoch. Zusammenfassend zeigt die vorliegende Arbeit die vielfältigen Funktionen von BCAA-basierten kleinen Molekülen sowie von BCAA-Transportern in der Interaktion zwischen dem S. aureus-Wirt und -Bakterium auf. Diese Arbeit liefert eine Konsolidierung der Rolle des AusAB NRPS in der S. aureus Lungeninfektion, gibt einen Einblick in die einzigartige Wirkweise des NRPS und schreibt dem NRPSProdukt Phevalin eine neuartige Funktion in der bakteriellen Kommunikation zu, die meines Wissens das erste Beispiel eines an der bakteriellen Kommunikation beteiligten natürlichen Monoketopiperazins darstellt. Darüber hinaus deuten Experimente mit ex vivo menschlichem Lungengewebe zum ersten Mal darauf hin, dass das AusAB NRPS an der Virulenz von Staphylokokken gegenüber menschlichem Lungengewebe beteiligt sein könnte. Zusätzlich beschreibt diese Arbeit einen möglichen Mechanismus für das Langzeitüberleben von S. aureus in Makrophagen, der eine BCAA-Eisenmetabolismus-Achse beinhaltet.

Staphylococcus aureus causes nosocomial- and community- acquired infections. This versatile pathogen expresses virulence factors (VF) that enhance establishment of infection and immune evasion. Our research focused on defining the roles of S. aureus VF on host immune responses during intracellular or extracellular infections. Accessory gene regulator (agr) controls VF expression and intracellular survival. Our goal was to determine mammary epithelial cells (MEC) responses to intracellular infection and subsequent polymorphonuclear leukocyte (PMN) responses. Intracellular S. aureus increased thrombomodulin expression by MEC and activated protein C (APC) production. APC inhibited PMN chemotaxis. Findings depicted an indirect role for VF on PMN responses, so next we determined signaling pathways and cytokine responses of PMN to S. aureus toxins. Live S. aureus infections increased activation of stress signaling pathways and highlighted a role for agr-regulated genes in MAPK p38 phosphorylation and α-hemolysin in ERK phosphorylation and IL-8 expression in PMN. Continuing our studies of VF, chemotaxis inhibitory protein of S. aureus (CHIPS) inhibits monocyte chemotaxis. We hypothesized that CHIPS inhibited C5a receptor (C5aR) signaling. Monocytes pretreated with CHIPS did not inhibit C5aR signaling. Nevertheless, signaling pathways can reduce PMN function in models such as glucocorticoid treatment. Immunosuppressive effects of glucocorticoids on PMN are restored with OmniGen-AF® supplementation. Glucocorticoid receptor and Toll-like receptor signaling potentially crosstalk to restore PMN function. OmniGen-AF® supplementation restored dexamethasone-induced immunosuppression in a MyD88-dependent manner. Overall, this research focused on characterizing immune responses to S. aureus infections and PMN signaling pathways and how it is key to understanding pathogenesis. / Ph. D.

<i>Staphylococcus aureus</i> is a versatile opportunistic pathogen causing a wide spectrum of diseases in both humans and animals. My research focused on characterization of the immune responses of monocyte derived dendritic cells (DC) to <i>S. aureus</i>. We initially evaluated the potential of circulating monocytes to serve as precursors for DC during <i>S. aureus</i> infection. The CD14⁺ monocytes, when stimulated with irradiated (ISA) or live <i>S. aureus</i> (LSA), differentiated into CD11c^high CD11b^high DC (MonoDC) in an autocrine fashion. This was associated with the up- regulation of granulocyte-macrophage colony stimulating factor (GMCSF) and tumor necrosis factor-α (TNF-α) gene transcription. We continued our studies to identify the role of TNF-α in the LSA induced differentiation of monocyte to MonoDC. Blocking TNF-α reduced the expression of CD11c and increased the expression of CD14 on LSA stimulated monocyte derived MonoDC. Stimulated monocytes were able to secrete monocyte chemotactic protein-1 (MCP-1), a chemokine that recruits monocytes to the site of infection/injury and induces the expression of β₂ integrins on DC. Characterization of the response of DC derived from monocytes using GMCSF and IL-4 revealed that, intact <i>S. aureus</i> rather than its purified structural components were efficient in DC activation. In response to ISA or LSA stimulation, DC induced proliferation of T cells collected from the peripheral circulation of cows with a history of <i>S. aureus</i> mastitis. Subsequent characterization of the proliferating T cells identified the presence of memory T cells. Finally, we identified a unique population of DEC205⁺CD8^a+ in monocyte derived DC. We further elucidated the role of DC DEC205, a C-type lectin, in <i>S. aureus</i> uptake. Blocking of receptor mediated endocytosis resulted in reduced uptake of <i>S. aureus</i> by DC. Confocal microscopy confirmed a role for DEC205 in <i>S. aureus</i> internalization and delivery to endosomes. DEC205 DC upon stimulation with <i>S. aureus</i> displayed enhanced maturation and antigen presentation. In conclusion, monocyte derived DC can uptake <i>S. aureus</i> and elicit cell mediated immune responses. / Ph. D.